A triazolothiadiazole compound, a preparation method and application thereof

Abstract

The present application relates to a kind of triazolothiadiazoles compound and its preparation method and application, belong to biological medicine technical field.The triazolothiadiazoles compound of the present application is structured as wherein, R1 is heterocyclic amino;R2 is selected from aryl or heteroaryl.This triazolothiadiazoles compound has significant inhibitory activity to ferroptosis inhibitor 1, and has inhibitory effect on the proliferation of HT1080 cell, with good antitumor effect;Illustrate that this triazolothiadiazoles compound can be used for preparing the disease related to ferroptosis inhibitor 1 mediation, and has potential application space in the development of antitumor drug.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a triazolothiadiazole compound and a preparation method and application thereof. BACKGROUND

[0002] Programmed cell death has multiple ways, including apoptosis, pyroptosis, autophagy, etc. Ferroptosis is a new non-apoptotic programmed cell death mode caused by excessive accumulation of iron-dependent lipid peroxides discovered in 2012. This cell death mode shows that when the free iron in the cell increases, on the one hand, more active oxygen will be produced through Fenton reaction to oxidize the polyunsaturated fatty acids on the cell membrane into lipid peroxides; on the other hand, the lipid peroxides can be further catalyzed into toxic lipid free radicals, resulting in cell death. Ferroptosis is found to be highly related to human diseases, such as neurodegenerative diseases, tissue damage during cold exposure, ischemia-reperfusion injury and cancer, etc. Inducing ferroptosis is a very promising method for treating malignant tumors, which shows a synergistic effect with cancer immunotherapy, and can even kill drug-resistant and metastatic tumor cells.

[0003] Since ferroptosis has become a very promising target for treating cancer, it has attracted great interest. So far, it has been found that ferroptosis is at least regulated by three major redox-related systems, including cysteine-glutathione (GSH)-glutathione peroxidase 4 (GPX4) system, guanosine triphosphate ring hydrolase-tetrahydrobiopterin (BH4) system and ferroptosis inhibitory protein 1 (FSP1)-CoQ 10 system. Among them, FSP1-CoQ 10 inhibits ferroptosis in cooperation with GSH-GPX4. FSP1 inhibits ferroptosis by reducing ubiquinone to ubiquinol, or reducing vitamin K to hydroquinone. Studies have shown that by inhibiting FSP1, the reducing capacity of FSP1 on lipid peroxides can be affected, thereby inducing the occurrence of cell ferroptosis and effectively preventing tumor recurrence, so the development of FSP1 inhibitors is of great significance for cancer treatment.

[0004] Although FSP1 inhibitors show great prospects in tumor treatment, there are still certain challenges in developing FSP1-targeted small molecule inhibitors at present, not only in terms of quantity, but also in terms of no FSP1 inhibitors reported in clinical research stage. The reported FSP1 inhibitors mainly include NPD4928, iFSP1, icFSP1, FSEN1, FSEN3, etc. These reported FSP1 inhibitors have shown the potency of inducing ferroptosis in several cancer cell lines, but their inhibitory effect on FSP1 is not strong enough, and their pharmacokinetic properties are poor, which is not suitable for in vivo use; in addition, they will exhibit off-target effects at high concentrations, so there is an urgent need for new FSP1 inhibitors with improved pharmacokinetic properties and pharmacodynamic characteristics to fully explore the potential of FSP1 as a tumor treatment target. SUMMARY

[0005] To this end, the technical problem to be solved by the present application is to overcome the problems of small selection range, insufficient inhibitory effect and poor drug property of FSP1 inhibitors in the prior art.

[0006] To solve the above technical problems, the present application provides a triazothiadiazole compound and a preparation method and application thereof.

[0007] The first object of the present application is to provide a triazothiadiazole compound, the structure of which is shown in formula V:

[0008]

[0009] wherein R1 is a heterocyclic amino group;

[0010] R2 is selected from aryl or heteroaryl.

[0011] In an embodiment of the present application, the heterocyclic amino group is selected from

[0012] the aryl group is selected from: the heteroaryl group is

[0013] In an embodiment of the present application, the triazothiadiazole compound is selected from the following compounds:

[0014]

[0015] The second object of the present application is to provide a preparation method of the triazothiadiazole compound, comprising the following steps:

[0016] S1, the compound of formula I, carbon disulfide and potassium hydroxide are reacted in a first organic solvent to obtain a compound of formula II;

[0017] S2, the compound of formula II is reacted under the action of hydrazine hydrate to obtain a compound of formula III;

[0018] S3, the compound of formula III and chloroacetic acid are reacted under the action of phosphorus oxychloride to obtain a compound of formula IV;

[0019] S4, the compound of formula IV and a secondary amine H-R1 are reacted in a second organic solvent under the action of N,N-diisopropylethylamine to obtain the triazolothiadiazole compound of formula V;

[0020] wherein the structures of formula I to formula V are as follows:

[0021]

[0022] wherein R1 is a heterocyclic amino group;

[0023] R2 is selected from aryl or heteroaryl.

[0024] In an embodiment of the present application, in S1, the first organic solvent is selected from one or more of methanol, ethanol, dichloromethane and ethyl acetate.

[0025] In an embodiment of the present application, in S2, the reaction temperature is 115-125°C and the reaction time is 16-24h.

[0026] In an embodiment of the present application, in S3, the reaction temperature is 95-115°C and the reaction time is 8-12h.

[0027] In an embodiment of the present application, in S4, the reaction temperature is 85-95°C and the reaction time is 10-14h.

[0028] In an embodiment of the present application, in S4, the second organic solvent is selected from one or more of acetonitrile, diethyl ether, dichloromethane and ethyl acetate.

[0029] In an embodiment of the present application, the reaction formula of the triazolothiadiazole compound is as follows:

[0030]

[0031] A third object of the present application is to provide a stereoisomer and / or a pharmaceutically acceptable salt of the triazolothiadiazole compound.

[0032] A fourth object of the present application is to provide a pharmaceutical composition, the active ingredient of which is the triazothiadiazole compound, the stereoisomer and / or pharmaceutically acceptable salt of the triazothiadiazole compound.

[0033] A fifth object of the present application is to provide the use of the triazothiadiazole compound, the stereoisomer and / or pharmaceutically acceptable salt of the triazothiadiazole compound, and the pharmaceutical composition in the preparation of a drug for treating FSP1 related diseases.

[0034] In an embodiment of the present application, the FSP1 related diseases include one or more of gastric cancer, prostate cancer, pancreatic cancer, breast cancer, ovarian cancer, lung cancer, hepatocellular carcinoma, melanoma, lymphoma, leukemia, copper tolerance, severe acute pancreatitis, diabetes, Parkinson's disease, retinal degeneration, age-related macular degeneration, acute kidney injury, early brain injury, liver fibrosis, and non-alcoholic fatty liver disease.

[0035] The technical solution of the present application has the following advantages compared with the prior art:

[0036] (1) The present application first constructs a compound system with a triazothiadiazole as the core skeleton. Compared with thiazolotriazoles FSP1 inhibitors, the replacement of the electronic isostere of the thiazole ring produces a unique spatial topology. The nitrogen atom at position 2 forms a specific hydrogen bond interaction with the Lys355 residue in the FSP1 catalytic domain. Compound P3 also establishes a T-shaped π-π stacking interaction with Tyr296 through the 4-(piperazine-1-acyl) methyl benzoate side chain, increasing the binding capacity of the compound to FSP1 and enhancing the inhibitory activity of FSP1.

[0037] (2) The triazothiadiazole compound of the present application can enhance RSL3 induced ferroptosis in different tissue-derived cancer cell lines, and target inhibits FSP1, blocking the regeneration pathway of CoQ 10 , VK class redox active molecules, thereby promoting the occurrence of phospholipid peroxidation and further promoting RSL3 induced cell ferroptosis. This indicates that the triazothiadiazole compound can be used as a sensitizer for combined therapy with GPX4 inhibitors, providing a new chemical entity for solving the problem of tumor drug resistance.

[0038] (3) The triazothiadiazole compound of the present application has significant inhibitory activity on ferroptosis inhibitor 1 and inhibitory effect on the proliferation of HT1080 cells, and has good antitumor effect. This indicates that the triazothiadiazole compound can be used for the preparation of treating ferroptosis inhibitor 1 mediated related diseases, and has potential application space in the development of antitumor drugs. BRIEF DESCRIPTION OF DRAWINGS

[0039] In order to make the content of the present application more easily understood, the present application will be further described in detail below according to specific embodiments of the present application and in conjunction with the accompanying drawings, in which:

[0040] Figure 1 Predicted binding mode and interaction analysis of compound P3 and FSEN3 in Test Example 1 of the present application; wherein, A is the potential binding mode of FSEN3 and FSP1 (PDB ID: 8WIK), B is the three-dimensional close-up view of the intermolecular interaction of compound P3 and FSP1 (residues around the ligand are white, carbon atoms of the ligand are painted orange, hydrophobic interactions are indicated by gray dotted lines, hydrogen bonds are indicated by green dotted lines, and π-π interactions are indicated by yellow dotted lines), C and D are two-dimensional interaction diagrams of FSEN3 and compound P3 with FSP1;

[0041] Figure 2 Test results of the compound enhancing RSL3-induced ferroptosis in Test Example 2 of the present application; wherein, A-C are cell survival rates of the indicated cancer cell lines treated with the indicated concentration of RSL3 in the presence of 1 μM of the indicated FSP1 inhibitor, and D is a representative image of A549 cells treated with 2.5 μM RSL3 in the presence of 1 μM of the indicated FSP1 inhibitor or DMSO;

[0042] Figure 3 Test results of the compound enhancing RSL3-induced lipid peroxidation in Test Example 3 of the present application; wherein, A-C are lipid peroxidation assessments of the indicated cancer cell lines treated with 2.5 μM RSL3 or DMSO in the presence of 1 μM of the indicated FSP1 inhibitor or DMSO, **p < 0.01, ****p < 0.0001, ns, no significance;

[0043] Figure 4 In Test Example 4 of the present application, HT1080 cells were treated with 2 μM RSL3, and the effect of VK on cell survival rate was observed. DETAILED DESCRIPTION

[0044] The present application will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present application and implement it, but the embodiments are not limiting of the present application.

[0045] In the present application, unless otherwise specified, the technical and scientific terms used in the present application are the same as the meanings commonly understood by those skilled in the art to which the present application belongs.

[0046] In the present application, unless otherwise specified, the term "and / or" used in the present application includes any and all combinations of one or more related listed items.

[0047] In the present application, unless otherwise stated, the experimental methods used in the embodiments of the present application are conventional methods, and the materials, reagents, etc. used, unless otherwise stated, can be obtained from commercial channels.

[0048] Example 1

[0049] The triazolothiadiazole compound of the present application and the preparation method thereof specifically include the following steps:

[0050]

[0051] S1, Synthesis of 4-amino-5-(4-methoxyphenyl)-2,4-dihydro-3H-1,2,4-triazole-3- thione (IIIa): Compound Ia (1.66 g, 10 mmol) and potassium hydroxide (1.12 g, 20 mmol), carbon disulfide (1.2 mL, 20 mmol) were added to ethanol at room temperature, and the reaction was carried out at room temperature for 12 h; TLC monitoring showed that the reaction was completed, and then ether was added, filtered, washed with water and ether, and dried to obtain white solid IIa (2.52 g, 90%); Compound IIa (2.52 g, 9 mmol) was dissolved in water, and 85% hydrazine hydrate solution (4.36 mL, 90 mmol) was added dropwise under stirring, and the reaction was carried out at 120°C for 20 h; TLC monitoring showed that the reaction was completed, and then water was added for dilution, and concentrated hydrochloric acid was added for acidification, and then cooled for crystallization, filtered, washed with water, dried, and recrystallized with ethanol to obtain white solid IIIa (1.0 g, 50%).

[0052] 1 H NMR (400 MHz, DMSO) δ 13.84 (s, 1H), 7.97 (d, J = 8.9 Hz, 2H), 7.08 (d, J = 9.0 Hz, 2H), 5.77 (s, 2H), 3.82 (s, 3H).

[0053] 13 C NMR (101 MHz, DMSO-d6) δ 166.6, 160.9, 149.4, 129.6, 118.1, 114.0, 55.4.

[0054] HRMS (ESI) m / z: [M+H] + calculated for C9H 10 N4OS: 223.0648; found: 223.0653.

[0055] S2, Synthesis of 6-(chloromethyl)-3-(4-methoxyphenyl)-[l,2,4]triazolo[3,4- b][l,3,4]thiadiazole (IVa): Compound IIIa (4.44 g, 20 mmol) and chloroacetic acid (2.27 g, 24 mmol) were added to phosphorus oxychloride at room temperature and heated to 100 °C for 10 h; the reaction was monitored by TLC and upon completion, quenched with cold water, neutralized with 40% NaOH solution, extracted with ethyl acetate, the organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate and concentrated in vacuum. The crude product was purified by silica gel column chromatography eluting with dichloromethane and methanol (dichloromethane: methanol = 50: 1) to afford IVa (2.24 g, 40%) as a yellow solid.

[0056] 1 H NMR (400 MHz, CDC13) δ 8.24 (d, J = 8.9 Hz, 2H), 7.04 (d, J = 8.9 Hz, 2H), 4.86 (s, 2H), 3.88 (s, 3H).

[0057] 13 C NMR (101 MHz, Chloroform-d) δ 165.6, 161.3, 154.0, 146.5, 128.0, 117.8, 114.3, 55.4, 39.6.

[0058] HRMS (ESI) m / z: [M+H] + calculated for C 11 H9ClN4OS: 281.0258; found: 281.0262.

[0059] S3, Synthesis of 6-(dihydroindol-l-ylmethyl)-3-(4-methoxyphenyl)- [l,2,4]triazolo[3,4-b][l,3,4]thiadiazole (P1): Compound IVa (0.2 mmol, 56 mg) was dissolved in dichloromethane (2 mL) at room temperature, dihydroindole (0.4 mmol, 45.7 μί) and N,N-diisopropylethylamine (0.8 mmol, 139 μί) were added and then the reaction was heated to 90 °C for 12 h; upon completion, the reaction was directly concentrated under reduced pressure and the crude product was purified by silica gel column chromatography eluting with petroleum ether and ethyl acetate (petroleum ether: ethyl acetate = 1 : 1) to afford P1 (57.4 mg, 79%) as a yellow solid.

[0060] 1H NMR (400 MHz, CDC13) δ 8.27 (d, J = 8.9 Hz, 2H), 7.18 (d, J = 7.2 Hz, 1H), 7.12 (t, J = 7.7 Hz, 1H), 7.05 (d, J = 8.9 Hz, 2H), 6.83 (t, J = 7.4 Hz, 1H), 6.58 (d, J = 7.8 Hz, 1H), 4.58 (s, 2H), 3.89 (s, 3H), 3.55 (t, J = 8.2 Hz, 2H), 3.10 (t, J = 8.2 Hz, 2H).

[0061] 13 C NMR (101 MHz, CDC13) δ 171.0, 161.2, 154.1, 150.6, 146.2, 130.0, 127.9, 127.5, 125.1, 120.1, 118.3, 114.3, 107.7, 55.4, 54.8, 50.9, 28.7.

[0062] HRMS (ESI) m / z: [M + H] + calculated for C 19 H 17 N5OS: 364.1227; found: 364.1224.

[0063] Example 2

[0064] The procedure was essentially the same as Example 1, except for S3, which comprises the following steps:

[0065]

[0066] S3, Synthesis of 5-((3-(4-methoxyphenyl)-[l,2,4]triazolo[3,4-b][l,3,4]thiadiazol-6- yl)methyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridine (P2): Compound IVa (0.2 mmol, 56 mg) was dissolved in dichloromethane (2 mL) at room temperature, 4,5,6,7- tetrahydrothieno[3.2-c]pyridine (0.4 mmol, 48.6 μί) and N,N-diisopropylethylamine (0.8 mmol, 139 μί) were added at room temperature, then the reaction was warmed to 90 °C and refluxed for 12 h; after the reaction was completed, the reaction solution was directly concentrated under reduced pressure, and the crude product was separated by silica gel column chromatography, eluted with petroleum ether and ethyl acetate (petroleum ether: ethyl acetate = 2: 1) to obtain yellow solid P2 (71.0 mg, 90%).

[0067] 1H NMR (600 MHz, CDC13) δ 8.26 (d, J = 8.9 Hz, 2H), 7.13 (d, J = 5.1 Hz, 1H), 7.04 (d, J = 8.8 Hz, 2H), 6.74 (d, J = 5.1 Hz, 1H), 4.07 (s, 2H), 3.88 (s, 3H), 3.82 (s, 2H), 3.01 (t, J = 5.6 Hz, 2H), 2.96 (t, J = 5.7 Hz, 2H).

[0068] 13 C NMR (151 MHz, CDC13) δ 172.1, 161.1, 154.3, 146.1, 133.0, 132.4, 127.9, 125.0, 123.3, 118.3, 114.3, 56.4, 55.4, 53.2, 50.8, 25.0.

[0069] HRMS (ESI) m / z: [M + H] + calculated for C 18 H 17 N5OS2: 384.0948; found: 384.0948.

[0070] Example 3

[0071] The procedure was essentially the same as Example 1, except for S3, which comprises the following steps:

[0072]

[0073] S3, Synthesis of methyl 4-(4-((3-(4-methoxyphenyl)-[l,2,4]triazolo[3,4- b][l,3,4]thiadiazol-6-yl)methyl)piperazin-l-yl)benzoate (P3): Compound IVa (0.2 mmol, 56 mg) was dissolved in dichloromethane (2 mL) at room temperature, 4-(piperazin-l-yl)benzoic acid methyl ester (0.4 mmol, 88.0 mg) and N,N-diisopropylethylamine (0.8 mmol, 139 μί) were added, then the reaction was warmed to 90 °C and refluxed for 12 h; after the reaction was completed, the reaction solution was directly concentrated under reduced pressure, and the crude product was separated by silica gel column chromatography, eluted with petroleum ether and ethyl acetate (petroleum ether: ethyl acetate = 1:1.5) to give white solid P3 (80.1 mg, 86%).

[0074] 1H NMR (400 MHz, CDC13) δ 8.26 (d, J = 8.9 Hz, 2H), 7.94 (d, J = 9.0 Hz, 2H), 7.04 (d, J = 8.9 Hz, 2H), 6.89 (d, J = 9.0 Hz, 2H), 3.95 (s, 2H), 3.88 (s, 3H), 3.87 (s, 3H), 3.45 - 3.38 (m, 4H), 2.84 - 2.82 (m, 4H).

[0075] 13 C NMR (151 MHz, DMSO-d6) δ 172.7, 166.1, 160.7, 154.0, 153.7, 145.0, 130.7, 127.4, 118.3, 118.0, 114.6, 113.5, 56.2, 55.4, 52.3, 51.5, 46.7.

[0076] HRMS (ESI) m / z: [M+H] + calculated for C 23 H 24 N6O3S: 465.1704; found: 465.1704.

[0077] Example 4

[0078] Example 1 except that S1, compound Ia is replaced by benzhydrazide Ib to give 6- (dihydroindol-1-ylmethyl)-3-phenyl-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole (P4);

[0079]

[0080] 1 H NMR (600 MHz, CDC13) δ 8.34 (d, J = 7.0 Hz, 2H), 7.54 (t, J = 7.4 Hz, 2H), 7.50 (t, J = 7.3 Hz, 1H), 7.19 (d, J = 7.3 Hz, 1H), 7.12 (t, J = 7.7 Hz, 1H), 6.84 (t, J = 7.4 Hz, 1H), 6.58 (d, J = 7.8 Hz, 1H), 4.59 (s, 2H), 3.56 (t, J = 8.2 Hz, 2H), 3.10 (t, J = 8.2 Hz, 2H).

[0081] 13C NMR (151 MHz, CDCI3) δ 171.4, 154.6, 150.5, 146.3, 130.3, 130.0, 128.9, 127.5, 126.3, 125.6, 125.1, 120.1, 107.7, 54.9, 50.9, 28.7.

[0082] HRMS (ESI) m / z: [M + H] + calculated for C 18 H 15 N5S: 334.1121 ; found: 334.1119.

[0083] Example 5

[0084] Example 1 except that S1, compound Ia was replaced with 4-chlorobenzhydrazide Ic to give 3-(4-chlorophenyl)-6-(dihydroindol-1 -ylmethyl)-[1,2,4]triazolo[3,4- b][1,3,4]thiadiazole (P5);

[0085]

[0086] 1 H NMR (400 MHz, CDCI3) δ 8.29 (d, J = 8.6 Hz, 2H), 7.52 (d, J = 8.6 Hz, 2H), 7.19 (d, J = 7.1 Hz, 1 H), 7.12 (t, J = 7.5 Hz, 1 H), 6.84 (t, J = 7.5 Hz, 1 H), 6.57 (d, J = 7.7 Hz, 1 H), 4.59 (s, 2H), 3.56 (t, J = 8.2 Hz, 2H), 3.11 (t, J = 8.1 Hz, 2H).

[0087] 13 C NMR (151 MHz, CDCI3) δ 171.8, 154.7, 150.5, 145.4, 136.3, 130.0, 129.2, 127.6, 127.5, 125.1, 124.1, 120.2, 107.7, 54.9, 50.9, 28.7.

[0088] HRMS (ESI) m / z: [M + H] + calculated for C 18 H 14 ClN5S: 368.0731 ; found: 368.0731.

[0089] Example 6

[0090] Example 1 except that in S1, compound Ia was replaced by 2-furoylhydrazine Id to give 3-(furan-2-yl)-6-(dihydroindol-1-ylmethyl)-[1,2,4]triazolo[3,4- b][1,3,4]thiadiazole (P6);

[0091]

[0092] 1 H NMR (400 MHz, CDC13) δ 7.68 (d, J = 1.8 Hz, 1H), 7.25 (d, J = 4.0 Hz, 1H), 7.18 (d, J = 7.3 Hz, 1H), 7.12 (t, J = 7.1 Hz, 1H), 6.83 (t, J = 7.4 Hz, 1H), 6.62 (dd, J = 3.4, 1.8 Hz, 1H), 6.57 (d, J = 7.8 Hz, 1H), 4.58 (s, 2H), 3.55 (t, J = 8.2 Hz, 2H), 3.10 (t, J = 8.2 Hz, 2H).

[0093] 13 C NMR (101 MHz, CDC13) δ 172.3, 153.9, 150.5, 147.3, 144.6, 140.7, 129.9, 127.6, 125.1, 120.2, 111.7, 111.6, 107.7, 54.9, 50.9, 28.7.

[0094] HRMS (ESI) m / z: [M+H] + calculated for C 16 H 13 N5OS: 324.0914; found: 324.0911.

[0095] Inhibition activity of test example 1 compounds on FSP1

[0096] Enzymatic reactions were prepared using TBS buffer (50 nM Tris-HCl and 150 nM NaCl) containing the molecule to be tested P1-P6 with 50 nM non-myr-FSP1, 200 nM NADH, using FSE N3 as positive control; then 100 μΜ of fluoroacetate sodium salt (Sigma, cat. no. R7017) was added to the reaction system, and the fluorescence intensity (F) was measured every minute at 37°C using a SpectraMax iD5 microplate reader and SoftMax Pro v.7 software, with an excitation / emission wavelength of 540 / 590 nm; IC50was calculated using equal volumes of DMSO and control reactions without fluoroacetate. 50Values; after the experiment, curve fitting and IC 50 Values were calculated, and the results are shown in Table 1:

[0097] Table 1 Inhibitory activity of compounds on FSP1 protein

[0098] Compound IC 50 (μM) Compound IC 50 (μM) Compound IC 50 (μM) P1 0.05±0.02 P2 0.18±0.05 P3 0.035±0.016 P4 0.09±0.07 P5 0.07±0.00 P6 0.19±0.06 FSEN3 0.15±0.09

[0099] As can be seen from Table 1, the triazolothiadiazole compounds of the present application have good inhibitory activity on FSP1, and the FSP1 inhibitory activity of compounds P2 and P6 is comparable to that of FSEN3, and the FSP1 inhibitory activity of compounds P1, P3, P4 and P5 is better than that of FSEN3.

[0100] The best enzyme inhibitory activity compounds P3 and FSEN3 were selected for molecular docking with FSP1, and compounds P3 and FSEN3 both used similar binding modes, and the predicted binding modes are shown in Figure 1 As can be seen from Table 1, the triazolothiadiazole compounds of the present application have good inhibitory activity on FSP1, and the FSP1 inhibitory activity of compounds P2 and P6 is comparable to that of FSEN3, and the FSP1 inhibitory activity of compounds P1, P3, P4 and P5 is better than that of FSEN3. Figure 1 As can be seen from Table 1, the triazolothiadiazole compounds of the present application have good inhibitory activity on FSP1, and the FSP1 inhibitory activity of compounds P2 and P6 is comparable to that of FSEN3, and the FSP1 inhibitory activity of compounds P1, P3, P4 and P5 is better than that of FSEN3.

[0101] Effect of test example 2 compounds on cell viability by inhibiting FSP1 combined with RSL3

[0102] FSP1 inhibitors can enhance RSL3-induced ferroptosis in cancer cell lines of different tissue origins, such as human non-small cell lung cancer cell line A549, human fibrosarcoma cell line HT1080 and human osteosarcoma cell line U2OS. Therefore, these three tumor cell lines were selected to determine whether the compounds can enhance RSL3-induced ferroptosis. A549, HT1080, U2OS cells were cultured to a cell density of about 80%, the cells were digested and centrifuged, and the cells were resuspended and counted using 1 mL of fresh culture medium, and then the cells were seeded into a 96-well plate at a density of 5000 cells / well, and 24 wells on the upper and lower edges of the plate were not seeded with cells, and were filled with 200 μL of PBS; the 96-well plate was placed in a cell incubator for culture, and after about 12 h, the cells were completely adherent, drug treatment was performed, and 1 μM of test compounds P1, P3, and FSEN3 were added to all wells as positive controls; after adding the test compounds, RSL3 was set to a concentration gradient of 0, 0.01 μM, 0.03 μM, 0.05 μM, 0.1 μM, 0.3 μM, 0.5 μM, 1 μM, 3 μM, 5 μM, 10 μM, and was added to the plate in turn, with 2 replicate wells for each concentration; after adding RSL3 and test compounds, cell death was observed, and after about 5 h, the 96-well plate was removed, and the cells in the plate were lysed using a CellTiter Glo kit, mixed thoroughly, and the fluorescence value was measured on a microplate reader (TECAN) to quantify the ATP level of the cells, and the relative viability was evaluated by ATP reading; data processing was performed using GraphPad, and the effect of the test compounds on cell viability by inhibiting FSP1 combined with RSL3 was calculated, and the results are shown in Table 1. Figure 2 As can be seen from Table 1, Figure 2 compounds P1 and P3 can significantly promote RSL3-induced ferroptosis in human non-small cell lung cancer cell line A549, human fibrosarcoma cell line HT1080 and human osteosarcoma cell line U2OS.

[0103] Effect of test example 3 compounds on cell lipid peroxidation by inhibiting FSP1 combined with RSL3

[0104] Since FSP1 exerts anti-ferroptosis activity by reducing RTA molecules such as CoQ or VK, and CoQ and VK can inhibit the propagation stage of phospholipid peroxidation to inhibit phospholipid peroxidation, the inhibition of FSP1 will theoretically promote the occurrence of phospholipid peroxidation. Based on this, the intracellular lipid ROS level was detected by flow cytometry. A549, HT1080, and U2OS cells were cultured to a cell density of about 80%, the cells were digested and centrifuged, and the cells were resuspended and counted using 1 mL of fresh culture medium, and then the cells were seeded into a 24-well plate at a density of 20,000 cells per well; the 24-well plate was placed in a cell incubator for culture, and after about 12 h, the cells were completely adhered, drug treatment was performed, the control group wells were added with corresponding concentration of DMSO, and the experimental group wells were added with 2.5 μM RSL3 (inducing ferroptosis) and 1 μM of the test compound P1, P3; after adding RSL3 and the test compound, the cell death was observed, and after about 5 h, the final concentration of 5 μM BODIPY 581 / 591C11 dye was added, and the cells were cultured at 37°C for 30 min; the 24-well plate was taken out, the cells were digested and centrifuged, washed once with HBSS, then resuspended in HBSS, and transferred to a flow tube; after mixing well, the signal from the oxidation probe was counted by FITC channel on a flow cytometer (BD FACS verse) to determine the intracellular lipid peroxidation level, and at least 5 x 10 3 cells per sample were analyzed; Flowjo was used for data processing to calculate the effect of the test compound on cell lipid peroxidation by inhibiting FSP1 combined with RSL3, and the results are shown in Figure 3 . As can be seen from Figure 3 , when the cells are co-incubated with compounds P1, P3 and RSL3, the content of lipid peroxide is significantly increased compared with cells treated with RSL3 alone, indicating that compounds P1 and P3 can enhance RSL3-induced lipid peroxidation.

[0105] Effect of test example 4 compound on HT1080 cell activity by inhibiting FSP1

[0106] It is assumed that the addition of a lethal amount of RSL3 in cells can induce ferroptosis, and if VK, a substrate of FSP1, is added at this time, FSP1 can provide a constant supply of VKH2 by reducing VK, so that tumor cells can resist the lethal amount of RSL3. In order to prove this hypothesis, 2 μM RSL3 was added to HT1080 cells to induce ferroptosis, and at the same time, a gradient concentration of VK was added, and the results are shown in Figure 4 . As can be seen from Figure 4It can be seen that about 0.3 μM VK can make the cells survive. The VK inhibiting ferroptosis depends on the reducing capacity of FSP1 in the cells to VK. If the reducing of VK is inhibited by exogenously adding FSP1 inhibitor in the case of adding 2 μM RSL3 and 0.3 μM VK to make the cells survive, the cells will die because of the inability of VK to be reduced. Using this system, the IC50 of the FSP1 inhibitor to FSP1 at the cell level can be determined by adding different concentration gradients of the FSP1 inhibitor. 50 It can also be proved that the inhibitor can specifically target FSP1.

[0107] The HT1080 cells were cultured to a cell density of about 80%, the cells were digested and centrifuged, and the cells were resuspended and counted using 1 mL of fresh culture medium, and then the cells were inoculated into a 96-well plate at a number of 5000 cells per well. The 24 wells on the upper edge and lower edge of the 96-well plate were not inoculated with cells because the evaporation rate of the culture medium in the edge wells of the 96-well plate was faster, and 200 μL of PBS was used to make up; the 96-well plate was placed in a cell incubator for culture. After about 12 h, the cells were completely adherent, drug treatment was performed, and 2 μM of RSL3 (inducing ferroptosis) and 0.3 μM of vitamin K (inhibiting ferroptosis, vitamin K is reduced and regenerated by endogenous FSP1 in the cells to provide a continuous supply of reduced vitamin K, i.e. VKH2) were added to all the wells; after adding RSL3 and vitamin K, the test compounds P1, P3-P5 and the positive control drugs iFSP1, FSEN3 were set to 12 different concentration gradients, and were sequentially added to the wells, with 2 replicate wells for each concentration; after adding the compounds, the cell death was observed, and after about 7 h, the 96-well plate was taken out, the cells in the wells were lysed using the CellTiter Glo kit, and the cell ATP level was quantified by measuring the fluorescence value on the enzyme label instrument (TECAN) after thorough mixing, and the relative survival ability was evaluated by the ATP reading value; the data were processed using GraphPad to calculate the IC50 of the test compounds and the positive control drugs, and the results are shown in Table 2: 50

[0108] Table 2 Proliferation inhibition results of compounds on HT1080 cells

[0109] Compound IC 50 (μM) Compound IC 50 (μM) Compound IC 50 (μM) P1 0.4215 P3 0.3404 P4 0.7517 P5 0.9776 FSEN3 0.7322 iFSP1 0.8493

[0110] It can be seen from Table 2 that the compounds P1 and P3 of the present application have a significant inhibitory effect on the proliferation of HT1080 cells, and the compounds P4 and P5 also have a certain inhibitory effect on the proliferation of HT1080 cells. It is proved that the triazolothiadiazole compounds of the present application have good antitumor effect and have broad application space as FSP1 inhibitors.

[0111] ​Obviously, the above embodiments are merely example for clearly illustrating, and are not limitation to the embodiments. For ordinary skilled in the art, other different forms of changes or variations can be made on the basis of the above description. Here, all the embodiments need not and can not be exhausted. The obvious changes or variations derived therefrom are still within the scope of the present invention.

Claims

1. A triazolothiadiazole compound or a pharmaceutically acceptable salt thereof, characterized by, The triazothiadiazole compound is selected from the following compounds:

2. The method of preparing a triazothiadiazole compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein The method comprises the following steps: S1, reacting a compound of formula I, carbon disulfide and potassium hydroxide in an organic solvent to obtain a compound of formula II; S2, reacting the compound of formula II under the action of hydrazine hydrate to obtain a compound of formula III; S3, reacting the compound of formula III and chloroacetic acid under the action of phosphorus oxychloride to obtain a compound of formula IV; S4, reacting the compound of formula IV and a secondary amine H-R1 in an organic solvent under the action of N,N-diisopropylethylamine to obtain the triazothiadiazole compound of formula V; The structures of formula I to formula V are as follows: The triazothiadiazole compound of formula V is the triazothiadiazole compound of claim 1.

3. A process for the preparation of a triazothiadiazole compound or a pharmaceutically acceptable salt thereof according to claim 2, characterized in that, In S1, the organic solvent is selected from one or more of methanol, ethanol, dichloromethane and ethyl acetate.

4. A process for preparing a triazothiadiazole compound or a pharmaceutically acceptable salt thereof according to claim 2, characterized by, In S4, the organic solvent is selected from one or more of acetonitrile, diethyl ether, dichloromethane and ethyl acetate.

5. A pharmaceutical composition, characterized by, The active ingredient of the pharmaceutical composition is the triazothiadiazole compound of claim 1 or a pharmaceutically acceptable salt thereof.

6. Use of the triazothiadiazole compound of claim 1 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition of claim 5 in the preparation of a medicament for treating a FSP1 related disease.

7. Use according to claim 6, characterized in that, The FSP1 related disease is selected from one or more of gastric cancer, prostate cancer, pancreatic cancer, breast cancer, ovarian cancer, lung cancer, hepatocellular carcinoma, melanoma, lymphoma, leukemia, severe acute pancreatitis, diabetes, Parkinson's disease, retinal degeneration, age-related macular degeneration, acute kidney injury, early brain injury, liver fibrosis and non-alcoholic fatty liver disease.

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